The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
Transfection processes are used to deliver various types of materials into a cell, and there are numerous known methods in the art. For example, U.S. Pat. No. 5,586,982 describes a treatment device capable of delivering genetic material or drugs into cells of a patient in vivo using heat to assist with transfection. Unfortunately, such approach often tends to damage the cells. Moreover, since the poration lasts only for a very short time, the amount of material delivered into the cells will in most cases be significantly reduced, especially where the material is relatively large. Finally, such approach also fails to provide a method for culturing cells after transfection.
In another example, as described in US 2009/0081750, magnetic fields are employed to move cells through a channel in which the cells undergo transfection. Actual transfection is then performed via several possible manners, including electroporation, heat, or light. Similar to the '982 reference, effective transfection is typically limited to relatively small molecules and low quantities. Yet another example of poration to transfect cells is described in WO 2013/059343. Here, cells are fed through a microfluidic channel in a buffer that contains a delivery material. The cells pass through a constriction region, which causes the cells to become perturbed with pores through which the delivery material then diffuses. While this approach overcomes to at least some degree issues associated with short pulse time, delivery still requires poration.
A more extreme approach is presented in U.S. Pat. No. 5,858,663 in which a cold gas shockwave is used to accelerate micro projectiles that carry matter into the cells. While such approach guarantees delivery of even relatively large molecules into a cell, it is readily apparent that such approach is also prone to significantly damage a cell.
WO 96/24360 attempts to overcome shockwave damage by providing a time-dependent impulse transient characterized by rise time and magnitude that is thought to increase the overall permeability of a cell membrane, which results in an increase in diffusion of materials into the cell. The impulse is achieved by applying an optical field to a film on which the cells are grown, and the optical field ablates the film thereby delivering the impulse. While such approach will provide for transfection, high throughput production of transfected cells remains problematic. To increase throughput, WO 02/42447 teaches use of leverages shock or other forms of pressure, and U.S. Pat. No. 7,687,267 describes a high throughput cell transfection device for transfer of small nucleic acid molecules (e.g., DNA, siRNA) through electroporation where the device contains an array of cell transfection units. Similarly, US 2012/0244593 teaches a high throughput electroporation transfection device, which requires poration (i.e., electroporation) and diffusion to deliver the material.
Unfortunately, the known transfection devices require significant disruption to a cellular membrane to allow for greater diffusion of cargo material, which becomes especially difficult where the cargo material is relatively large. For example, clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has emerged as an important tool for performing targeted and highly-efficient editing of a cell's endogenous genome as evidenced by Cong, L. et al. “Multiplex Genome Engineering Using CRISPR/Cas Systems”, Science 339, 819-823 (2013); and Ran, F. A. et al. “Genome engineering using the CRISPR-Cas9 system”, Nature Protocols 8, 2281-2308 (2013). The two essential components of CRISPR technology are a guide RNA and a RNA-guided nuclease, e.g. Cas9. The guide RNA specifies the targeted DNA sequence while recruiting the Cas9 nuclease to the target site for gene editing. Advantageously, multiple genes can be targeted simultaneously by delivering a multiplex of different guide RNA sequences.
One especially desirable use of such complex gene editing is the generation of induced pluripotent stem (iPS) by co-expressing of a cocktail of transcription factors in differentiated somatic cells to so reprogram the cells into a pluripotent state (see e.g., Takahashi, K. & Yamanaka, S. “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”, Cell 126, 663-676 (2006)). Using mRNAs encoding the respective reprogramming factors, iPS cells can be generated without leaving a genetic footprint in the reprogrammed cells with higher efficiency compared to other DNA-based approaches as indicated by Warren, L. et al. “Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA”, Cell Stem Cell 7, 618-630 (2010); and Mandal, P. K. & Rossi, D. J. “Reprogramming human fibroblasts to pluripotency using modified mRNA”, Nature Protocols 8, 568-582 (2013).
However, transfection efficiency and viability using multiple distinct nucleic acids is often problematic and requires in all or almost all instances multiple steps during which cells need to be transferred or otherwise manipulated. As a consequence, currently known transfection methods often require significant time to generate iPS cells, and viability and/or yield is often much less than desired.
Therefore, there is a need for improved transfection devices and methods for delivery of cargo of various sizes, and especially large and/or multi-component cargo into a cell in a manner that will not or only minimally adversely affect the cell.